Not Applicable
The present invention relates generally to genetic sequence of plants that encode polyphenol oxidase (PPO) enzymes and functional fragments and parts thereof. More particularly, the present invention provides nucleic acid molecules encoding polyphenol oxidase enzymes of lettuce, banana, tobacco and pineapple plants. The invention further provides methods of isolating said nucleic acid molecules.
Browning of plant tissues often occurs following injury or damage and this generally results in spoilage of fruit and vegetables. Undesirable browning also occurs during processing of plant materials to produce food or other products. Steps are taken during transport, storage, and processing to prevent these browning reactions. Often this involves the use of chemicals such as sulphur dioxide but the use of these substances is likely to be restricted in the future due to concerns about their safety and consumer acceptance. For example, the US Food and Drug Administration banned the use of sulphite for most fresh fruit and vegetables in 1986. The production of fruit and vegetable varieties with an inherently low susceptibility to brown would remove the need for these chemical treatments.
It will be understood that browning in plants is predominantly catalysed by the enzyme PPO. PPO is localised in the plastids of plant cells whereas the phenolic substrates of the enzyme are store in the plant cell vacuole. This compartmentation prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substrates are mixed.
The prior art includes International Application PCT/AU92/00356 to the present applicant which describes the cloning of PPO genes from grapevine, broad bean leaf, apple fruit and potato tuber. This application recognises that PPO levels in plants may be manipulated by increasing or decreasing expression of PPO gene. The application also identifies two conserved copper binding sites in PPO genes, designated CuA and CuB. However, the method described in PCT/AU92/00356 which was used to clone the PPO genes from apple and potato involved the use of an oligo dT reverse primer for polymerase chain reaction (PCR). Whilst the method is acceptable, in some tissues, it does not give rise to a strong band of the predicted size or else it gives rise to many additional products making it difficult to resolve the PPO fragment.
Accordingly, it is an object of the present invention to overcome or at least alleviate one or more of the difficulties related to the prior art.
This application is a continuation-in-part application of continuation-in-part application of U.S. Ser. No. 08/976,222, filed Nov. 21, 1997, and International Application No. PCT/AU98/00362 filed May 19, 1998, the entire contents of which are incorporated herein by way of reference.
Bibliographic details of the publications referred to in this specification by author are collected at the end of the description.
Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification appear after the claims.
Throughout this specification and the claims that follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d will be understood to imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
In work leading up to the present invention, the inventors sought to produce improved methods for isolating PPO-encoding nucleic acid molecules which are susceptible for use in modifying the expression of endogenous PPO genes in plants, to reduce browning and modify ripening and storage characteristics of plant tissues and organs.
Accordingly, the inventors have cloned several PPO-encoding genes from lettuce, tobacco, banana and pineapple and produced recombinant gene constructs comprising same for the expression of recombinant PPO polypeptides and nucleic acids capable of modifying the PPO content of plant tissues and cells when expressed therein.
One aspect of the present invention provides an isolated nucleic acid molecule that comprises a nucleotide sequence which encodes or is complementary to a nucleotide sequence which encodes a PPO polypeptide of lettuce, banana, tobacco or pineapple having an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, or comprising the copper-binding site of any one of said amino acid sequences.
In an alternative embodiment, the present invention provides an isolated nucleic acid molecule that encodes a PPO polypeptide of lettuce, banana, tobacco or pineapple wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29;
(ii) a fragment of (i) comprising a nucleotide sequence that encodes the copper-binding site of a PPO polypeptide;
(iii) a degenerate nucleotide sequence of (i) or (ii); and
(iv) a nucleotide sequence that is complementary to (i) or (ii) or (iii).
A second aspect of the invention provides gene constructs comprising the isolated nucleic acid molecules of the invention, preferably in a format suitable for expression in plants, particularly in banana, lettuce, tobacco or pineapples.
A third aspect of the invention provides a method of modifying the endogenous PPO activity of plant cells, tissue, or organs, particularly those cells, tissues, and organs of lettuce, banana, tobacco and pineapples, by expressing the isolated PPO-encoding nucleic acid molecules, or a fragment or analogue or homologous thereof, in the sense or antisense orientation therein for a time and under conditions sufficient to modify transcription or translation of the endogenous mRNA encoding PPO and/or to produce a functional PPO enzyme. As used herein, the word xe2x80x9cmodifyxe2x80x9d clearly encompasses any alteration to a stated integer, including both a reduction and an increase thereof.
Accordingly, in one embodiment, this aspect of the invention provides a method of increasing the level of lettuce, banana, pineapple or tobacco PPO activity in a plant or a cell, tissue or organ thereof, said method comprising:
(i) introducing a nucleotide sequence to said plant or a cell, tissue or organ thereof which sequence encodes a PPO polypeptide of lettuce, banana, tobacco or pineapple having an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, or an enzymatically-active PPO polypeptide comprising the copper-binding site of any one of said amino acid sequences; and
(ii) expressing said nucleotide sequence to produce an enzymatically-active PPO polypeptide.
In an alternative embodiment, this aspect of the invention provides a method of increasing the level of lettuce, banana, pineapple or tobacco PPO activity in a plant or a cell, tissue or organ thereof, said method comprising:
(i) introducing a nucleic acid molecule to said plant or a cell, tissue or organ thereof which nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29, or a degenerate nucleotide sequence thereof; and
(ii) expressing said nucleic acid molecule to produce an enzymatically-active PPO polypeptide.
In an alternative embodiment, this aspect of the invention provides a method of decreasing the level of PPO activity in a plant or a cell, tissue or organ thereof, said method comprising introducing a nucleic acid molecule to said plant or a cell, tissue or organ thereof which comprises a nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence which encodes a PPO polypeptide of lettuce, banana, tobacco or pineapple having an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, or the copper-binding site of any one of said amino acid sequences;
(ii) a nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29;
(iii) a fragment of (ii) comprising a nucleotide sequence that encodes the copper-binding site of a PPO polypeptide; and
(iv) a nucleotide sequence that is complementary to (i) or (ii) or (iii).
A fourth aspect of the present invention clearly extends to transfected and transformed cells, tissues, organs and whole organisms that have the subject nucleic acid molecules of the invention introduced thereto. The introduced nucleic acid molecules may exist as extra chromosomal genetic material, or alternatively or in addition, in a form that has been integrated into the cellular genome. This aspect of the invention clearly encompasses transformed plants and plant parts and propagules comprising the subject nucleic acid molecules as an addition to their normal genome composition.
A further aspect of the invention relates to methods of isolating homologous of the nucleic acid molecules exemplified herein, in particular methods relying upon nucleic acid hybridization between highly-conserved regions of the exemplified sequences and nucleotide sequences of homologous PPO-encoding sequences. Such methods include standard nucleic acid hybridizations (i.e. RNA: DNA and RNA: RNA and DNA: DNA) and polymerase chain reaction (PCR)-based and isothermal amplification methods.
According to this aspect of the invention, there is provided a method for preparing nucleic acid encoding an internal fragment of a PPO polypeptide of banana, lettuce, tobacco or pineapple comprising at least a portion of a copper-binding site of said polypeptide or a hybridizable fragment of said nucleic acid, said method including:
(i) providing:
(a) banana, lettuce, tobacco or pineapple PPO cells, tissue or organs having PPO activity;
(b) a first primer having a nucleotide sequence capable of hybridizing to a copper (Cu) binding site-encoding region of a PPO gene or upstream thereof;
(c) a second primer having a nucleotide sequence capable of hybridizing to the complement of a copper (Cu) binding site-encoding region of a PPO gene or downstream thereof; and
(d) an adaptor primer;
(ii) isolating RNA from said cells, tissues or organs;
(iii) treating the RNA to construct copy DNA (cDNA) therefrom; and
(iv) amplifying the cDNA so formed using the first and second primers.
Preferably, the first primer comprises a nucleotide sequence selected from the group consisting of:
(i) 5xe2x80x2-GCGAATTCTT[TC][TC]TICCITT[TC][CA][TC][AC]G-3xe2x80x2(SEQ ID NO: 31);
(ii) 5xe2x80x2-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3xe2x80x2(SEQ ID NO: 32);
(iii) 5xe2x80x2-GCGAATTCAA[TC]GTIGA[TC][AC]GIATGTGG-3xe2x80x2(SEQ ID NO: 33);
(iv) 5xe2x80x2-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3xe2x80x2(SEQ ID NO: 34);
(v) 5xe2x80x2-GCGAATTCTTICCIT[TA][TC]TGGAA[TC]TGGG-3xe2x80x2(SEQ ID NO: 35); and
(vi) a hybridizable fragment of any one of (i) to (v).
Preferably, the second primer comprises a nucleotide sequence selected from the group consisting of:
(i) 5xe2x80x2-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3+(SEQ ID NO: 36);
(ii) 5xe2x80x2-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3xe2x80x2-(SEQ ID NO: 37); and
(iii) a hybridizable fragment of (i) or (ii).
Preferably, the treatment of RNA to construct cDNA is performed by treating the RNA with reverse transcriptase and an adaptor primer that comprises the nucleotide sequence:
5xe2x80x2-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3xe2x80x2(SEQ ID NO: 38) or a hybridizable fragment thereof to form cDNA.
Nucleic acid encoding the N-terminal fragment of the PPO polypeptide of banana, lettuce, tobacco or pineapple can be obtained by attaching an anchor to the 5xe2x80x2-end of the cDNA formed and amplifying said cDNA using a first primer that binds to said anchor and a second primer in the antisense orientation, wherein the nucleotide sequence of said second primer is derived from the sequence of the internal PPO fragment. In this embodiment, the primer in the sense orientation may comprise a nucleotide sequence selected from the group consisting of:
(i) 5xe2x80x2-ATATCACCTGTCGGTACATGACGGC-3xe2x80x2(SEQ ID NO: 39);
(ii) 5xe2x80x2-GTGCCATTGTAGTCGAGGTCAATCA-3xe2x80x2(SEQ ID NO: 40);
(iii) 5xe2x80x2-CCAGTGCCTGGTTTAGGTGTATTCAC-3xe2x80x2(SEQ ID NO: 41); and
(iii) a hybridizable fragment of (i) or (ii) or (iii).
Additionally, in a preferred embodiment, the primer in the antisense orientation may comprise a nucleotide sequence selected from the group consisting of:
(i) 5xe2x80x2TGCTGTTCTGTTCGAACATGGCAG-3xe2x80x2(SEQ ID NO: 42);
(ii) 5xe2x80x2-TATACAAGTGGCACCAGTGTCTGC-3xe2x80x2(SEQ ID NO: 43);
(iii) 5xe2x80x2-CCGCATTGTGGATGACTTCCATCTG-3xe2x80x2(SEQ ID NO: 44);
(iv) 5xe2x80x2-CCAGAATGGGATGGTGAAGGTGTCG-3xe2x80x2(SEQ ID NO: 45); and
(v) a hybridizable fragment of any one of (i) to (iv).
Nucleic acid encoding the C-terminal fragment of the PPO polypeptide of banana, lettuce, tobacco or pineapple can also be obtained by amplifying said cDNA using an adaptor primer and a primer in the sense orientation, wherein the nucleotide sequence of said second primer is derived from the sequence of the internal PPO fragment. In this embodiment, the primer in the sense orientation may comprise a nucleotide sequence selected from the group consisting of:
(i) 5xe2x80x2CGCTGGGTGGGTAATTCTAGGATG-3xe2x80x2(SEQ ID NO: 46);
(ii) 5xe2x80x2-AGTCATCCACAATGCGGCGCACATG-3xe2x80x2(SEQ ID NO: 47); and
(iii) 5xe2x80x2-GTTGCTCTTCTTAGGCTCGGCTTAC-3+(SEQ ID NO: 48)
(iv) a hybridizable fragment thereof.
The adaptor primer may include the following sequence or a hybridizable fragment thereof: 5xe2x80x2-GACTCGAGTCGACATCG-3+(SEQ ID NO: 49).